FAQs
Find answers to some of the most frequently asked questions about our Instruments & Software, Cell Lysis, ChIP, FFPE Nucleic Acid Extraction, and Nucleic Acid Shearing.
Topic
While some denaturing is tolerable, I have disulfide bonds in my protein that I do not want to disrupt. Will truXTRAC Protein Extraction Buffer DF leave disulfide bonds intact?
truXTRAC Protein Extraction Buffer DF has the presence of 8 M urea. This buffer will break the disulfide bond to a certain extent. You can always evaluate it with buffers such as SuperB which will help in maintaining the three-dimensional structures.
I am interested in extracting intact proteins for downstream functional assays. Functional assays involve a co-factor stain that can be used to check for protein presence by SDS-PAGE and if protein is strep tagged, it can be detected by anti-strep antibody on Western blot. Will truXTRAC® Protein Extraction Buffer SuperB affect this functional assay?
truXTRAC Protein Extraction Buffer SuperB has non-detergent sulfobetaine (NDSB) which helps in denaturing the protein. If you are interested in functional assays, please consider using Buffer N.
What non-ionic detergents work well with AFA?
- Triton X-100
- Octylglucoside (octyl-beta-D-glucopyranoside)
- NP-40
- Tween-20
Does Covaris provide an alternative type of detergent containing a buffer for LC/MS analysis?
The Covaris Protein Extraction Buffer DF is a detergent-free buffer that efficiently extracts proteins and is compatible with LC/MS analysis.
What is the best buffer to increase total protein yields?
Protein Extraction Buffer TP extracts more total protein at lower detergent concentrations in conjunction with AFA. This buffer contains urea, thiourea, and a zwitterionic detergent.
Describe the most suitable buffer for recovering proteins in native conformation?
Covaris Native Protein Extraction Buffer N is a physiological buffer optimized for extracting proteins in their native conformation and is directly compatible with immunoassay, protein assay, affinity enrichment, and any other applications requiring proteins in their native conformation.
What is the best buffer for extracting denatured proteins?
truXTRAC Protein Extraction Buffer TP or Buffer MJ are recommended for the extraction of denatured proteins.
Can I take extracted protein in Buffer TP or Buffer MJ for direct analysis by MS?
If you are doing LC/MS, then you may inject the sample directly. If you wish to perform reduction/alkylation followed by tryptic digestion, then you will want to dilute the protein to decrease the salt concentration.
Do native protein extraction buffers have any percent of organic compounds?
Both protein extraction Buffer N and SuperB are hydrophilic in nature and do not contain any organic compounds.
What is the advantage of using truXTRAC Protein Extraction Buffer SuperB instead of truXTRAC Protein Extraction Buffer N?
Protein Extraction Buffer SuperB increases protein yield and preserves the native conformation and biological activity. This buffer does not form micelles and prevents protein aggregation; however, protein Buffer N does have protein aggregation in low percentage.
How should I collect adherent cells and perform sample quantification?
There are a number of different methods which can be used to quantify the number of adherent cells. Typically, one can culture an additional plate, trypsinize the cells, followed by counting. The counts should be consistent with the other culture plates when starting at the same time.
Why do I need to run a fixation time course? What cell types is it most critical for and why?
Fixation is not only cell line dependent, but is also epitope dependent. Not all cells fix at the same rate, therefore, it is imperative to carry out both a one-time fixation and shearing time course to establish a protocol for cell lines and tissue types. Furthermore, certain proteins can be quite sensitive to formaldehyde fixation resulting in conformational changes from over-fixation that render them unrecognizable by an antibody.
Why is the methanol-free formaldehyde provided with truChIP kits recommended over other fixing solutions?
Many formaldehyde reagents contain methanol as a stabilizing agent to prevent oxidation and polymerization. Based on our evaluations, we have shown that the presence of methanol leads to the generation of large molecular weight chromatin complexes which is resistant to shearing.
Why is the fixation time in the Covaris truChIP ® protocol so short?
In stark contrast to Covaris AFA, bath and probe sonicators utilize up to 150× more energy. As a result, these other methods demand longer fixation times in order to minimize damage to the chromatin. Covaris AFA is a more efficient and gentle mechanical shearing technology that does not require long fixation times.
Why do we need to run a shearing time course if we have already done ChlP before?
Unfortunately, not all cells shear to the same size range at the same rate. As a result, Covaris recommends users to carry out a shearing time course to empirically determine the optimal treatment conditions. For ChIP-seq, it is important to optimize the chromatin shearing size range to be compatible with the selected library preparation kit and sequencing platform. For Illumina platforms, it is recommended to shear chromatin to an average fragment size of 200-250 bp with a distribution of up to ~700 bp.
What type of cells can truChIP kit be used for?
Covaris truChIP kits have been successfully tested with hundreds of mammalian cell lines by laboratories across the world. The truChIP kits are considered universal sample preparation kits guaranteed to work with all mammalian cells. The truChIP kits provide easy optimization for new cell lines or epitopes of interest. Learn more about truChIP kits in the related protocols
What are the lowest cell inputs that have been tested?
The truChIP Ultra-low Chromatin Shearing Kit (Covaris PN: 520156) has been used with ∼10,000 cells.
I want to process 50M cells/sample–is it possible?
Yes, our milliTUBE–1 mL with AFA fiber (PN: 520135) can accommodate up to 30 million cells. Additionally, one can use the milliTUBE–2 mL (PN: 520132) for samples with higher inputs up to 60 million cells. Note: The milliTUBE–2 mL is only compatible with the S220 and E220 platforms.
Does Covaris provide a protocol for tissue ChIP?
Yes, Covaris provides a truChIP kit (PN: 520237) for processing tissue masses within the range of 20-125 mg.
Is it possible to fragment native ChIP using a Covaris?
Because the energy can be tightly controlled, native ChIP sample prep can be performed using AFA. Please contact our Application’s team for further guidance.
Can you simultaneously lyse and fragment in one tube?
Generally, it is not possible to lyse and shear chromatin at the same time in one tube. One-step methods require high energy processing with high detergent concentrations. The yield of those methods tends to be significantly lower than two-step methods. For very low cell numbers, it is possible to do a one-step lysis and shearing with our truChIP ultra low cell chromatin shearing kit.
May I use my own buffers and reagents?
Most chromatin sample preparation and shearing buffers were developed and optimized for use with bath or probe sonicators. Since AFA is a more advanced ultrasonic technology, off-label buffers are not compatible with AFA. Therefore, we strongly recommend customers to use the buffers provided with the truChIP kit since they have been validated using Covaris instruments.
May I combine your protocol with my other commercial protocols?
The truChIP kits are compatible with downstream IP protocols. Covaris provides the formulation of the buffers to ensure the IP step can be performed with any homebrew or commercial IP kit. For example, the truChIP chromatin shearing reagent kit shearing buffer for use with cells contains 1 mM EDTA, 10 mM Tris-HCl pH 7.6, 0.1% SDS.
How does having reduced SDS concentrations help with the Covaris kit?
Reduced SDS concentrations enables the utilization of more sheared chromatin in each IP. Additionally, SDS is a potent solubilizer. Shearing with high concentrations of SDS can solubilize a significant amount of the desired epitope.
Is there a required RNase treatment?
Covaris recommends RNase treatment since the presence of RNA can potentially skew the smear analysis performed on the Bioanalyzer and agarose gels.
Why am I not seeing a gradual reduction in fragment size distribution as I shear my samples?
There are a few possibilities why no shearing is observed:
- The cells were processed using reagents and protocols not optimized for use with AFA
- The cells are being severely over-fixed
- The crosslinked sheared chromatin has not been reversed or purified properly for DNA size range analysis
I optimized protocols for a cell line and now am observing inconsistent shearing results. Why?
To ensure consistent results are achieved, Covaris recommends using the truChIP kit and to keep the cell numbers within the range outlined in the protocol. Any modifications made to the buffers and/or reagents provided, or deviations in the protocol, may lead to inconsistent shearing profiles.
Why did my IP fail?
IP can fail for a myriad of reasons. Some of the most common ones are:
- Over shearing resulting in epitope damage
- Fixation was not optimized for the protein of interest–the crosslinking was insufficient
- Incorrect IP buffer that has not been optimized for use with the selected antibody
Is it true that the fixation time cannot be reduced if a target of interest is not abundant?
No, the target abundance is independent of formaldehyde fixation. There is no direct correlation between fixation time and epitope abundance.
I have a large protein complex. What methods are recommended for these sample types?
For protein complexes, depending on the proximity of the protein of interest to the DNA, Covaris recommends the use of a dual fixation strategy. Specifically, this involves fixing proteins to proteins first with DSS, DGS, and DMA followed by fixing the proteins to DNA with formaldehyde.
If I fix it in less/time, will I lose some downstream sensitivity?
Since fixation is cell line and epitope dependent, it is certainly possible that the fixation might be too little or too much which can affect IP efficiency. Thus, this is why it is essential to carry out dual time course studies. Additionally, we recommend running a Western blot to check the epitope integrity during the shearing time course.
What is the average processing time per sample using truChIP?
Unfortunately, not all cells shear to the desired size using the same treatment time. Therefore, Covaris recommends that you carry out a fixation and a shearing time course to determine the optimal conditions. All truChIP protocols provide the details required to perform the optimization runs.
Why do I need to control the temperature?
Formaldehyde fixation is reversible with temperature. If temperature is not controlled during chromatin shearing, undesired reverse crosslinking may occur which leads to epitope loss.
Specifically, how does the duty factor (DF), power, and duration impact the fragment length?
For chromatin shearing using truChIP, Covaris has already optimized the duty factor (DF), peak incidence power (PIP), and cycles per burst (CPB). Therefore, the user is only responsible for optimizing the shearing time for the cell line or tissue sample.
May I use the truChIP kit with the M220 focused ultrasonicator?
Yes, all Covaris Focused-ultrasonicators are compatible for use with the truChIP kits.
Are larger processing volumes available?
Yes. For processing volumes greater than 1 mL, Covaris provides the milliTUBE–2 mL which can be used to shear chromatin in 2 mL volumes on the S and E-series instruments only. With these tubes, one can double the number of cells and volume. For sample treatment settings, the duty factor (DF) will be doubled (compared to 1 mL protocol) and the processing times will increase.
What settings should I use on the Covaris for ChIRP/Hi-C, etc.?
ChIRP and Hi-C protocols have not been optimized in-house, but we do have customers who are using it. Please contact our Application’s team for further guidance.
Where can I find protocols for the cryoPREP® Extraction Systems?
Protocol is published on our website. Click here to visit the protocols page and filter by cryoPREP.
What impact levels are recommended with cryoPREP (CP02) Impactor for my tissue samples?
Impact level varies and will depend on both the tissue type and input mass. Typically, Covaris recommends using level 5 or 6 for hard tissues such as skin, blood vessels, cornea, retina, cartilage, and bone. We recommend using level 2 to 4 if processing soft tissues.
For more information on the recommended impact levels, refer to the chart below:
| TT05/TT05M/ TT05XT/TT05MXT | TT1/TT1XT | TT2 | |||
|---|---|---|---|---|---|
| 20-80 mg | 80-300 mg | 300-500 mg | 500-1000 mg | 1000-2000 mg | |
| Brain | 1 | 2 | 3 | 4 | 6 |
| Liver | 2 | 3 | 3 | 4 | 6 |
| Kidney | 3 | 4 | 4 | 6 | 6 |
| Smooth Muscle | 4 | 4 | 4 | 6 | 6 |
| Skeletal/Cardiac Muscle | 5 | 5 | 5 | 6 | 6 |
| Ocular Tissue | 5 | 6 | 6 | 6 | 6 |
| Vascular Tissue | 5 | 6 | 6 | 6 | 6 |
| Skin | 6 | 6 | 6 | 6 | 6 |
| Tumor | 5 | 5 | 5 | 6 | 6 |
| Bone | 6 | 6 | 6 | 6 | 6 |
| Cartilage | 6 | 6 | 6 | 6 | 6 |
Can I impact the sample in the tissueTUBE more than once?
The tissueTUBEs TT05, TT05M, and TT1 are designed to withstand one impact. If a second impact is required, the extra thick (XT) version of each tissueTUBE is recommended. The extra thick bags can be impacted more than once. After the first impact, carefully inspect the tissueTUBE for any punctures prior to refreezing in liquid nitrogen and before its second impact.
What is the working mass range of the different tissueTUBEs?
Please refer to the table below for the recommended impact levels according to their mass ranges:
| TT05/TT05M/ TT05XT/TT05MXT | TT1/TT1XT | TT2 | |||
|---|---|---|---|---|---|
| 20-80 mg | 80-300 mg | 300-500 mg | 500-1000 mg | 1000-2000 mg | |
| Brain | 1 | 2 | 3 | 4 | 6 |
| Liver | 2 | 3 | 3 | 4 | 6 |
| Kidney | 3 | 4 | 4 | 6 | 6 |
| Smooth Muscle | 4 | 4 | 4 | 6 | 6 |
| Skeletal/Cardiac Muscle | 5 | 5 | 5 | 6 | 6 |
| Ocular Tissue | 5 | 6 | 6 | 6 | 6 |
| Vascular Tissue | 5 | 6 | 6 | 6 | 6 |
| Skin | 6 | 6 | 6 | 6 | 6 |
| Tumor | 5 | 5 | 5 | 6 | 6 |
| Bone | 6 | 6 | 6 | 6 | 6 |
| Cartilage | 6 | 6 | 6 | 6 | 6 |
Can I add a buffer to my tissue in the tissueTUBE before processing with the cryoPREP CP02 Impactor?
No, it’s important for tissue samples to remain dry for optimal cryofracture efficiency.
If residual tissue remains on the walls of the tissueTUBE after transferring the pulverized material, what buffer is recommended to collect remaining material from the tissueTUBE for nucleic acid extraction?
Any aqueous buffer utilized in RNA extraction/purification workflow can be used to transfer the residual tissue remaining in the tissueTUBEs.
If residual tissue remains on the walls of the tissueTUBE after transferring the pulverized material, what buffer can I use to transfer from tissueTUBE for protein extraction?
Acetonitrile, Methanol Ethanol, DMF, NMP, Isopropanol and Chloroform, as well as any aqueous buffers used in protein extraction/purification workflows.
Can cryoPREP Extraction Systems be used for plant material?
Yes, any agricultural material or biological material can be pulverized using the Covaris cryoPREP.
Once the sample is pulverized, is it mandatory for the sample to be processed immediately, or can it be stored?
The sample is not required to go through sample processing immediately. After cryofracturing, tissueTUBE plugs can be used to seal the tissueTUBEs for long-term storage at -80°C, or with vapor phase liquid nitrogen. Alternatively, the cryofractured tissue can be transferred into cryovials for long-term storage.
Can cryofractured tissue be freeze/thawed multiple times?
Multiple freeze/thaw cycles are never recommended for the extraction of biomolecules. We recommend long-term storage of cryofractured tissues in small aliquots to avoid multiple freeze/thaw cycles.
Can the samples in the tissueTUBE thaw?
Yes, this is a possibility if the tissue samples are not maintained at the necessary working temperatures either before or after cryofracturing.
Does the frozen tissue after cryo-impact still remain frozen through the whole process of fracturing?
Yes, prior to cryofracturing, we advise keeping the tissueTUBE/transfer tube assembly on dry ice. Tissue samples in the tissueTUBE are immersed in liquid nitrogen for 30 to 60 seconds to ensure efficient cryofracturing of the tissue samples. After CP02 impact, the tissue is quickly transferred into the transfer tubes and can be stored on dry ice, vapor phase liquid nitrogen, or for long-term storage at -80° C until use.
Does the process result in lysed/fractured cells or is it possible to fix the resulting frozen powder in formaldehyde to recover intact cells for FACS sorting?
Cryofracturing of fresh tissue damages the cell membrane; therefore, recovering significant numbers of intact cells after cryofracturing is not possible.
What are the reasons why the cryofractured material is not completely pulverized?
Certain tissues such as high adipose content tissues will “cake” immediately after impact rather than cryofracturing into distinct powder. Therefore, we recommend for the “caked tissue” to be quickly disrupted into smaller pieces inside the tissueTUBE prior to transfer into the transfer tube. Some other reasons for inefficient cryofracturing are:
- Tissue mass is too large for the tissueTUBE being used, or the impact level is too low for the tissue mass being used. Please refer to the impact level and tissue mass chart.
- Tissue is not centered in the tissueTUBE during impact.
- Liquid nitrogen incubation time is too short. We recommend a minimum of 30 seconds for tissue masses less than 100 mg, and 60 seconds for tissue mass of up to 2 g.
Can cryoPREP CP02 Impactor impact pig skin (0.5mm thickness)?
Yes, the CP02 can pulverize the pig skin at 0.5mm thickness into a fine homogenate when treated properly in liquid nitrogen.
Why has the tissueTUBE broken open during cryofracture?
There are various reasons why the tissue TUBES break open, these include:
- Tissue mass was too large for the tissueTUBE being used
- Tissue was not centered in the tissueTUBE. Tissues expand during impact, and if placed close to the seams, it will burst the seams of the tissueTUBE.
- Tissue was not properly flash frozen in liquid nitrogen. The key is to submerge the sample in liquid nitrogen for 30 to 60 seconds depending on the mass of the tissue.
- Previously frozen tissues will have sharp edges which can pierce through the tissueTUBEs. In such cases, we recommend an initial low impact level of 1 or 2, followed by another treatment with liquid nitrogen, and another impact at 4-6 depending on the tissue type.
- When processing tissues in tissueTUBEs TT05 and TT1, it is important to loosen the transfer tube by a quarter turn to allow venting of the rapidly expanding liquefied air.
What is the cryoPREP (CP02) Impactor and its key benefits?
CP02 is an electronically controlled cryofracturing instrument which consistently delivers the same amount of impact force to the samples. As a result, this provides very efficient, reproducible, and high sample-recovery results when compared to the manual processes such as bead beating or mortar and pestle.
What accessories and/or consumables do I need to process my tissue samples?
Click here for the correct combinations depending on your sample type and mass period.
When would I use standard tissueTUBE or extra thick tissueTUBE (XT)?
If you are processing hard or sharp tissue types, then the extra thick tissueTUBEs (XT) are recommended.
Can I store my samples in tissueTUBE?
Yes, samples may be stored in a tissueTUBE before pulverizing and after pulverizing by utilizing the appropriate plugs to seal the tissueTUBEs for long-term storage.
At what temperature can the tissueTUBEs be stored?
tissueTUBEs can be stored at -80° C, on dry ice, or in vapor phase liquid nitrogen.
What material is the tissueTUBE material made of?
It is made of DuPont Kapton Polyimide material.
What is the dimension of a piece after pulverization?
After cryofracture, the sample is a fine powder, or may turn into flat dough which can then be crumbled into a fine powder.
Can cryofractured tissue be stored at -80°C in the glass Covaris transfer tubes?
We do not recommend storing samples in glass tubes at -80° C. Please transfer to appropriate cryotubes for long-term storage.
On what AFA instruments can the cryofractured samples be treated for biomolecules extraction?
Depending on the biomolecule being extracted, all Covaris platforms can support your extraction workflows. Please contact Covaris Application’s Support for more details.
Are service plans for the cryoPREP (CP02) Impactor offered?
The CP02 is provided with a one year warranty at the time of the purchase. Additional years of service coverage are available for purchase. Please consult your territory sales manager for additional details.
Where can I find protocols for FFPE nucleic acid extraction?
A list of our current truXTRAC FFPE extraction protocols are published on our website. Click here to visit our Protocols webpage to find the protocol that is related to your specific requirements.
Can I extract RNA or total nucleic acids using truXTRAC FFPE kits?
Yes, truXTRAC FFPE kits are available for RNA and total nucleic acids. For further information, visit our page for truXTRAC FFPE Kits.
Can I extract proteins using truXTRAC FFPE kits?
Covaris and ProtiFITM have partnered to address challenges encountered when performing protein extraction from archived FFPE tissues. The partnership led to the development of a simple, efficient, and optimized proteomics workflow enabled by Covaris and ProtiFI. The Covaris Adaptive Focused Acoustics® (AFA®) technology combined with the Covaris truXTRAC capabilities drastically improves the sample prep by actively removing more paraffin while rehydrating tissue to isolate proteins prior to purification. ProtiFi, a protein purification technology, developed the S-TrapTM columns to trap AFA-released proteins and digest them prior to analysis.
Are the truXTRAC FFPE kits xylene-free?
Yes, our Covaris Adaptive Focused Acoustics technology® (AFA®) allows for active paraffin removal in aqueous buffer, compared to the less efficient passive paraffin removal, which uses organic solvents.
What tissue sample types can be used?
FFPE sections mounted on slides, scrolls or cores are compatible with the truXTRAC FFPE protocols.
What is the maximum sample size for use with FFPE truXTRAC protocols?
The protocols are optimized for sections up to 25 µm in thickness and cores up to 1.2 mm in diameter.
What collection tools are recommended for sampling slide-mounted FFPE sections?
Covaris recommends using a scalpel or razor blade to scrape material from slides. Please contact applicationsupport@covaris.com if you are experiencing difficulties.
Why is my DNA/RNA yield low? How can I improve my yield?
Low yield might be caused by several different factors such as:
- Low tissue to wax ratio in FFPE section: Excess paraffin will adversely affect the yield and quality of DNA and RNA extracted from FFPE tissue. We strongly advise trimming off any excess paraffin before sectioning a FFPE tissue block, or after the section has been cut from the FFPE block. A ratio of 80% tissue to 20% paraffin, or higher, is ideal. Repeat the procedure using additional sections until desired yield is achieved. In your initial use of the truXTRAC FFPE kit, use FFPE blocks that have been well characterized for yield and quality.
- Insufficient tissue input: Increase FFPE tissue section thickness or use more sections up to 5mg total weight.
- Proteinase K stored above recommended temperature or expired: Repeat the procedure using fresh Proteinase K. Always store Proteinase K solution at -20°C after resuspension.
If using bead purification, low DNA yield may also be caused by:
- Incorrect number of beads were added: Ensure beads are resuspended well by vortexing beads thoroughly before each use.
- Binding was incomplete: Make sure the correct reagent volumes were used and mixed well before the 56°C incubation.
- Beads lost during pipetting steps: Slowly draw up the supernatant into the pipette tip without disturbing the beads. It is normal for beads to stick to the pipette tips during the elution step without having an effect on the yield.
Why do I have no DNA/RNA?
Ensure that the protocol steps have been closely followed, including ethanol addition to wash buffers and mixing of the Proteinase K with the sample on the Covaris instrument. Proteinase K buffer contains glycerol, and may fall to the bottom of the microTUBE, therefore, thorough mixing is essential.
The concentration of DNA/RNA is too low, what could be the cause?
The elution volume may be too high. Repeat the procedure using lower elution volume (50 µl minimum volume is required for DNA and 20 µl for RNA). Alternatively, you may concentrate your samples using ethanol precipitation or by other means.
When following the FFPE DNA protocol with bead purification I notice clumping of magnetic beads during wash steps. Is this a problem?
Some clumping of magnetic beads may occur with specific FFPE blocks. Bead clumping won’t affect the yield or purity of the DNA. If bead clumping is severe enough to clog the pipette tips, reduce the input sample amount.
Magnetic beads are sticking to the pipette tips during elution whilst following the FFPE DNA bead purification protocol. Will this affect my DNA yield?
Some sticking of magnetic beads to pipette tips may occur with specific FFPE blocks. Magnetic beads sticking to pipette tips during elution will not affect the yield or purity of the DNA. If bead sticking is severe enough to clog pipette tips reduce the input sample amount.
Why does my DNA not perform well in downstream applications such as qPCR?
DNA in FFPE sample blocks could be severely cross-linked or degraded, therefore, ensure amplicons are designed to be as small as possible (less than 100 bp). DNA isolated using Covaris AFA technology is of the highest possible quality. Some FFPE sample blocks may be too degraded or cross-linked for some applications.
The DNA fragment size is too large when following Option A of the FFPE DNA protocol. What is the cause of this?
Too much emulsified paraffin in the sample absorbs some of the acoustic energy and will adversely affect DNA shearing efficiency. Trim any excess paraffin from tissue blocks before proceeding with the protocol. We recommend running a time course for DNA shearing (Step 7) to increase the treatment time by 30 second steps.
Which Covaris Focused-ultrasonicators are compatible with truXTRAC FFPE protocols?
All Covaris AFA instruments are compatible with truXTRAC FFPE kits. However each instrument will require specific accessories which may need to be purchased separately. Please reference the protocol related to your kit to learn more about required accessories.
How do I convert a method developed on an S2 or E210 for use with SonoLab™ 7?
What happened to Intensity (as employed in previous versions of SonoLab)?
In SonoLab 5 or later, Intensity has been replaced by Peak Incident Power (PIP) to better represent acoustic treatment conditions. In the S2 and E210 instruments, Intensity served as a dimensionless proxy for the power emitted from the transducer during each acoustic burst, with Intensity 10 being equivalent to approximately 350 Watts of Peak Incident Power. New generation instruments operating under SonoLab 5, 6, 7, and 8 are capable of delivering up to 500 Watts of Peak Incident Power (depending on the instrument). Rather than to invent a new dimensionless scale, Covaris chose, in SonoLab 7, to specify Peak Incident Power in true Watts.
Why do I need to change the water in the tank every day?
Purity of the water in the bath is very important for the proper acoustic coupling. When applying acoustic energy in rate-limited applications, foreign material such as algae and particulates may scatter the high frequency focused acoustic beam, resulting in a shift to a larger mean fragment size. Bath water should be deionized water and changed daily unless continuously treated by the optional Covaris Water Conditioning System (available for S, E, and LE instruments). Click to learn more about the WCS System.
Besides the daily changing of water in the bath, what are the other maintenance recommendations?
For all instruments, except the M220, the water bath and the degassing lines should be rinsed once a month with a 10% bleach solution. Refer to the recommendations below for more guidance:
- Fill the bath with 10% bleach solution
- Lower the transducer and run the degassing pump for a few minutes
- Raise the transducer and empty the tank
- Lower the transducer and run the pump for 5-10 seconds to empty the lines
- Fill the water bath with fresh water and repeat the procedure
The ME220 bleach protocol is run using the fill bottle based on the procedure from the user manual.
I have the Covaris Water Conditioning System. What kind of maintenance should I perform?
The Water Conditioning System (WCS) automatically circulates water through a particulate filter and ultraviolet (UV) sterilizer to ensure that water remains clean and free from algae growth for up to a month. When the system runs continuously, it maintains the proper water quality required for optimum performance of the transducer. Without the WCS, proper water quality is maintained by daily changes of the water in the bath. On a monthly basis, the water tank should be cleaned with a bleach solution as described above.
IMPORTANT: Disconnect the hoses to and from the WCS when performing the bleaching procedure. DO NOT circulate the bleach solution through the WCS. The UV lamp and filter should be replaced once a year as indicated by the hour meter on the front of the WCS. Instructions for this can be found in the user manual.
Why is it so important to degas the water in the tank?
Water in the tank (not in the chiller) serves as a coupler in the acoustic circuit. The need for clean and degassed water cannot be overstated. Poor water quality may dramatically impede the high frequency energy transfer. Insufficiently degassed water will readily scatter acoustic energy. The same can happen when water is contaminated with small particles. Keep in mind, that at high frequency, the acoustic energy may be scattered by dissolved gases and vapors present in water at standard atmospheric conditions as well as by particles suspended in water. Note: Degassing is not required for the M or ME instruments.
Can I add algaecide to my water bath?
No, algaecide should not be added to the water in the tank to avoid formation of the aerosol containing algaecide during the AFA treatment. However, you can add algaecide to the water in the chiller since the water is circulating through a stainless steel loop immersed in the water bath.
When I am done using the Covaris system, how should I properly shut down the instrument?
To properly shut down the instrument, turn off the degassing then raise the acoustic assembly to lift the transducer out of the water, and empty the water bath. Place the tank back on the instrument, turn on the degas pump for 5 seconds to empty the remaining liquid, dry it with a lint-free cloth, and place it back into the system.
What is AFA-grade water?
It is water that meets the AFA-grade specifications (ASTM Type III or ISO Grade 3) such as M220 AFA-grade Water (800ml) (PN 520101). AFA-grade water is recommended for Covaris Focused-ultrasonicators to properly complete the acoustic circuit.
The chiller is for some reason taking over an hour to chill the water bath on my S220. What could be the cause?
There could be several reasons why the chiller doesn’t cool the water bath efficiently. First, you should check the fittings – are they firmly connected? Are the hoses free of kinks? Are the hoses being pinched? Have the length of the hoses been increased? Are there any particulates present in the chiller water that can cause clogging? Has the chiller set point been changed? Is the water (or fluid) level in the chiller adequate; low level results in inefficient chilling?
I have a VWR chiller that came with the Covaris instrument. I can’t change the temperature on the chiller control panel. What should I do?
The reason why the current settings cannot be changed is because the local lockout feature on the controller has been activated. The screen is reading, “LLo[QN1] .” To unlock the controls, press and hold the Select/Set Knob for 10 sec. The screen will read, “CAN,” and the setting can now be modified.
How can I convert a method from one model instrument to another?
There is not a standard way to transfer method settings between all instruments. The best resource for developing a method is to reference our current shearing protocols which is available here: For advanced questions or for applications not listed on the website, please contact applicationsupport@covaris.com.
How can I properly install Covaris instrument drivers on my laptop?
Instructions for driver installation are available in the instrument user manual. For earlier instrument models (S2 and E210), please contact techsupport@covaris.com
What is the latest version of SonoLab 6 software (for E and LE instruments)?
SonoLab 6.2.8 was the last version of SonoLab 6 software released. SonoLab 6 is technically obsolete and has been replaced by SonoLab 7.3. The SonoLab 7 platform features improved error recording and the addition of history files. The same settings are used for protocols in both versions of software. If you have questions or concerns about upgrading, please contact techsupport@covaris.com.
| Software Version | M220 | ME220 | S220 | E220 | LE220 | E220evo |
|---|---|---|---|---|---|---|
| 6.2.8 (can be replaced by 7.3.6.3) | X | X | ||||
| 7.2.1.0 | X | X | ||||
| 7.3.6.3 | X | X | X | |||
| 8.0.0.373 | X |
Can AFA water be cooled in advance to speed up the preparation time?
AFA water must be degassed prior to cooling. Degassing is very ineffective at colder temperatures. Covaris does not recommend chilling water in advance. The Water Conditioning System is an optional accessory that can be used to keep water clean so that it does not need to be changed more than once a month. It is available for the S, E, and LE series instruments. Please contact your local sales representative for more information.
How can I be sure that the degas pump is running?
The degas pump should be audible when running. Additionally, you can check for very small bubbles coming from the degas outlet tube. If bubbles do not appear to be coming out of the outlet tube, the intake tube may need to be cleaned. The intake tube has 6 very small holes where the water is pulled in. These can be cleaned by wiping down and using a narrow gauge wire to poke through any build up that is blocking the holes. If this does not solve the problem, your degas pump may need to be replaced. Please contact techsupport@covaris.com if you believe the pump is not working. M and ME instruments do not contain a degas pump.
Our facility is prohibited from using bleach. Is there an alternative that can be used?
Hydrogen peroxide is a substitute which can be used to clean the tank and internal fittings of the instrument. This should approximately be 1.5% hydrogen peroxide solution. In E/LE series instruments with a 5L water tank, this is 1L of 8% hydrogen peroxide which is the most commonly available concentration. For the S-Series water tank, 1/3 of this amount should be used.
If the maintenance or check flow light appears on my WCS, how do I turn this off?
The Maintenance light indicates that the filter and bulb need to be replaced (this is recommended annually). After replacement, the alert can be reset with the button inside the cover. See the WCS user manual for additional information.
The Check Flow light indicates that the pressure is building up and water flow is restricted. The hose connections may need to be reset if they are not seated properly. If this does not solve the problem, the filter may need to be replaced if it is blocked.
The Check Flow light indicates that the pressure is building up and water flow is restricted. The hose connections may need to be reset if they are not seated properly. If this does not solve the problem, the filter may need to be replaced if it is blocked.
Where can I find DNA shearing protocols?
Shearing protocols differ between instruments. The current DNA shearing protocols are published on our website.
What are the DNA fragment sizes I can get using Covaris Focused-ultrasonicator instruments?
Covaris instruments can provide tunable DNA fragment sizes from 150 bp to 5kbp. Random shearing with Adaptive Focused Acoustics® (AFA®) energy generates a log normal distributed DNA fragment profile. We provide protocols for the most commonly required DNA sizes. Click here to access all of our protocols or visit the specific instrument webpage. Typically, sizes between the defined protocols may be achieved by increasing the time of the treatment of the nearest size DNA fragment (upper side).
How can I shear genomic DNA into fragment sizes larger than 5 kb?
The single use g-TUBE can be used to shear DNA into large fragment sizes ranging from 6 kb to 20 kb using a benchtop centrifuge. Click here to learn more about the g-TUBE on the product page .
We have 300 bp DNA which we want to shear to 150 to 200 bp. Can we use the recommended protocol?
Random DNA shearing generates a log normal distribution of fragments. The smaller the size of the input DNA, an exponentially higher amount of energy is required to fragment it to the desired smaller size range. The relationship between size and energy is demonstrated in the figure below. When starting with an input sample containing large fragments of DNA, less energy is required to randomly shear those large fragments to a desired average fragment size. When starting with a smaller input fragment size, more energy is required to shear randomly and generate a distribution of fragments around a desired average fragment size.
Our recommended protocols for shearing genomic DNA as a starting material requires more than a tenfold larger input starting material than the desired fragment size. When the difference between the size of DNA starting material and desired fragment size is small, increasing the AFA treatment time, but maintaining all other AFA parameters is recommended. For example, on the M220 instrument using a microTUBE-130 to shear gDNA to 150 bp, treatment time is 330 sec. If using 300 bp DNA as the starting material for the same size, you will need to increase the shearing time beyond 330 sec. Please run a time course with 10% incremental steps, and check the results on the Bioanalyzer.
Relationship between DNA fragment sizes and total energy needed.
Our recommended protocols for shearing genomic DNA as a starting material requires more than a tenfold larger input starting material than the desired fragment size. When the difference between the size of DNA starting material and desired fragment size is small, increasing the AFA treatment time, but maintaining all other AFA parameters is recommended. For example, on the M220 instrument using a microTUBE-130 to shear gDNA to 150 bp, treatment time is 330 sec. If using 300 bp DNA as the starting material for the same size, you will need to increase the shearing time beyond 330 sec. Please run a time course with 10% incremental steps, and check the results on the Bioanalyzer.
Relationship between DNA fragment sizes and total energy needed.
I do not see any settings for the fragment size I am interested in, but I see settings for size ranges a bit over and a bit below what I am interested in. How can I optimize settings to get the size range of interest?
Look at the settings which brackets the DNA fragment size of interest. Take the treatment conditions for the larger fragment size and increase the treatment time until the desired fragment size is reached. Conversely, you can take treatment conditions for the smaller size fragment and decrease the treatment time.
Why does the size distribution of fragments seem to get wider with increasing size range?
The scale on an electropherogram generated by the Agilent Bioanalyzer DNA chip is logarithmic, hence it is the shape of the peak. A peak at 2000 bp, with the same base width in seconds as a peak at 500 bp, will cover much wider bp range because of the logarithmic scale. Below is a picture of electropherogram of the 500 bp DNA fragment and the same data shown using a linear scale.
I have an Illumina protocol for creating 400bp fragments that uses T6 round bottom glass tubes. What alternatives are recommended?
Covaris has developed DNA shearing protocols using a microTUBE-500 that generates an equivalent DNA fragment size distribution as the one generated in T6 tubes. These protocols are available for all Covaris focused-ultrasonicators to find all of the Covaris Focused-ultrasonicator protocols.
Aside from the phosphodiester bonds, are there any other bonds broken during processing of my samples with the Covaris AFA?
To date, there has not been a study to show the effect of acoustic fragmentation on the bonds within the DNA molecule. Indirect evidence from billions of bases sequences so far indicate to a great extent that only phosphodiester bonds are broken during AFA fragmentation of DNA, generating a population of blunt and overhangs of varying lengths which are easily repaired during the initial steps of library preparation.
In what buffer type can I shear my DNA?
We recommend Tris-EDTA pH 8.0 buffer (TE) to give consistent results for a broad range of DNA fragment sizes.
Can I use the Covaris Focused-ultrasonicator instrument to fragment RNA? If so, what conditions should I use?
Yes, it is possible to fragment RNA using Covaris AFA technology. To fragment mRNA or total RNA into 200 nt fragments click here to find all Covaris protocols and filter by your specific instrument.
Can I use the published protocols to shear PCR amplicons?
Yes, the PCR amplicons can be fragmented using standard protocols as long as the size difference between starting material and desired fragment size is tenfold. The treatment time needs to be adjusted when the size difference is smaller. We recommend running a time course experiment to determine the length of treatment required. As a rule of thumb, if the size of your starting material is within the size range of the generated DNA fragments, you need to extend the treatment time (e.g. if you are trying to get DNA fragments with a mean size of 200 bp the range of fragments that are generated is between 50 and 700 bp).
Does the DNA source have an effect on the shearing conditions and results?
The Covaris AFA random fragmentation is a DNA source independent process. The protocols on the Covaris web site were developed using human, lambda and E. coli genomic DNA. Shorter lengths of starting material may require a longer treatment time to reach the desired fragments sizes.
What is the minimum concentration of input DNA for the g-TUBE?
The g-TUBE has been tested with DNA input as low as 100 ng. For lower inputs, we recommend reducing centrifugation speed if necessary.
Can I use a microcentrifuge tube to shear the DNA?
No, the sample vial is a critical component of the acoustic circuit of the Covaris AFA process. The sample vessel material, thickness, geometry and the solution in the vessel all play an important role in this circuit. Following the Covaris recommended protocols and using the appropriate Covaris sample vessels will provide the optimal results. This flyer illustrates the dramatic effect on thermal control of using a microcentrifuge tube instead of a microTUBE.
Can I use microTUBE or miniTUBE for DNA storage?
No, the microTUBE or miniTUBE should not be used for storage, and samples should be transferred after processing.
I notice a fiber inside the microTUBE/milliTUBE. What is the purpose of this fiber, and should I be worried about contamination of my samples from this fiber during treatment?
The fiber inside the tubes serves a dual purpose. The first is providing nucleation sites for inertial cavitation. Cavitation is a process where a bubble in the liquid rapidly collapses creating a microjet that fragments the DNA. The second purpose is to allow for efficient mixing of the sample during processing. The acoustic fiber is thoroughly cleaned during the manufacturing process before being inserted inside the tubes and is free from organic contaminations.
Which centrifuges are compatible with the g-TUBE?
- Eppendorf MiniSpin plus
- Eppendorf Model 5415 R with temperature set at 30° C
- Eppendorf Model 5424
If you have another brand of centrifuge, please contact applicationsupport@covaris.com.
Why am I not getting consistent results when shearing DNA?
Inconsistent results might be caused by several different factors, however, there are few important things to check first:
- Sample volume: Each consumable has been optimized for a specific sample volume in order to allow optimal performance. For example, the presence of large headspace allows for the occasional formation of an air gap in the tubes, disrupting the consistent fragmentation of DNA to the desired size range. Click here to find instrument protocols which include instrument specific recommended sample vessels for different volumes.
- Water level: The water level is critical for DNA shearing. Not only does the water allow for the acoustic energy to couple from the transducer into the tube, it is also important in keeping your sample at the appropriate temperature during processing and minimizing vibrations which could lead to glass tube breakage. Exact water levels to follow are published for each instrument and consumable, click here for the latest information.
- Water bath temperature: Water temperature should be closely controlled and matched to the application. Warmer temperatures promote less forceful collapse of acoustic cavities within the sample fluid, causing a shift toward larger mean fragment size, therefore the water bath temperature during DNA shearing should be tightly controlled.
- Degas level of water: For S, E and LE- series instruments, insufficient degas levels within the bath may result in less efficient acoustic coupling and thereby shift the mean fragment size. System degas pumps should be run in advance and during AFA treatment. Prior to running a process the water bath should be degassed for at least 30 min to one hour, until the “degas” indicator in the SonoLab software can be checked without error.
- Water purity: Foreign materials such as algae and particulates may scatter the high frequency focused acoustic beam, resulting in a shift to larger mean fragment size. Bath water should be pure distilled or DI water, changed daily or cleansed by a Covaris Water Conditioning System.
I am not getting the expected DNA fragment sizes. How can I check whether the instrument is working properly?
To check whether the instrument is working properly, we recommend using the Covaris DNA Shearing Verification Kit (PN 520120). The user manual is available here for your reference DNA Shearing Verification Kit User Manual
When analyzing DNA fragments on the Bioanalyzer, I am noticing tailing or even a split peak. What is the probable cause?
Loading too much DNA onto a chip can distort the peak which causes a split or tailing to occur. The split peak or shoulder on the right hand side of the peak can also be caused by an occasional formation of an air gap in a microTUBE or milliTUBE. This will result in partitioning of the liquid within the tube and thus, the sample will not receive uniform acoustic treatment. As a result, larger DNA fragments are observed in the upper region of the electropherogram. To avoid the air gap formation, the correct sample volume for the tube type must be used and care should be taken to avoid introduction of air during pipetting. You can also use the Covaris Centrifuge and Heat Block microTUBE Adapter (PN: 500406) or the Centrifuge microTUBE Adapter 6x16mm (25) (PN 520059) to remove any air bubbles introduced during the pipetting of the sample into the microTUBE. This reusable adapter fits most benchtop microcentrifuges.
When analyzing DNA fragments on the Bioanalyzer, I am noticing a small peak near the lower marker. What is the probable cause?
The extra peak can be caused by the presence of RNA in the DNA preparation. The RNA can be sheared similar to DNA into small fragments. However, the single stranded RNA runs faster than DNA fragments of the same length. If you are performing PCR prior to shearing, the smaller peak might also indicate primer dimer formation, an artifact of the PCR reaction not generated by shearing.
I followed the settings provided in the DNA Shearing Quick Guide for 300bp, but my distribution is centered on 320 bp on a High Sensitivity Bioanalyzer chip. Why is this?
With the exception of the 320 µl microTUBE-500 protocol (High Sensitivity DNA Kit, cat# 5067-4626), mean DNA fragment size distributions published in Covaris DNA Shearing Guide are based on electropherograms generated from the Agilent Bioanalyzer with DNA 12000 Kit (cat# 5067-1509). DNA fragment representation will vary with analytical systems, therefore, please carry out a time course based on the settings provided in this document to reach the desired fragment size distribution. Please see for an illustration.
When I check my sheared samples on a gel, the smear profile looks different to the size range you indicated on your protocol. Why is that?
The smear profile on a gel is dependent on the concentration of the loaded DNA sample, as well as the staining technique that is used. Overloading the gel will give an impression of a wider distribution. Also, staining the gel after the electrophoresis will avoid the gradient formation of Ethidium Bromide.
What is the 96 AFA-TUBE TPX Plate made of?
The 96 AFA-TUBE TPX Plate is made of a unique polymer for low impedance and better transmission of acoustic energy. The material focuses and efficiently couples acoustic energy and optimizes cavitation. The 96 AFA-TUBE TPX Plate has also been designed for integration with common labware such as heat blocks, magnetic bead stands, and most thermal cyclers.
How can we achieve similar results in the 96 AFA-TUBE TPX Plate to the microTUBE if there is no AFA fiber?
The 96 AFA-TUBE TPX Plate uses an engineered polymer vessel that incorporates features which effectively controls acoustic cavitation to enable reproducible and precisely tuned hydrodynamic shearing forces.
What Covaris AFA systems can we use the 96 AFA-TUBE TPX Plate with?
Currently, the R230 and the LE220-plus is the only system validated to work with the 96 AFA-TUBE TPX Plate.
Can I use a heat seal with the 96 AFA-TUBE TPX Plate?
Heat sealing is not compatible with the 96 AFA-TUBE TPX Plate. Covaris provides a foil seal that has been optimized to work with the 96 AFA-TUBE TPX Plate.
Can I use an automated peeler?
We have not validated any automated peelers for use with the 96 AFA-TUBE TPX Plate.
Can I use an automated sealer?
Automated foil adhesive sealing with the Biosero KAPS 500 is compatible with the 96 AFA-TUBE TPX Plate.
Can I use other adhesive foils other than the one supplied by Covaris?
Additional adhesive foils have not been validated for the 96 AFA-TUBE TPX Plate.
What temperature can we incubate the 96 AFA-TUBE TPX Plate?
The 96 AFA-TUBE TPX Plate has been incubated up to 96°C with the Covaris foil seal and a heated lid. We do not recommend incubations above 25°C without a heated lid.
Can I store samples in the 96 AFA-TUBE TPX Plate?
Sample storage in the 96 AFA-TUBE TPX Plate has not been validated.
Why isn’t the 96 AFA-TUBE TPX Plate compatible with LE220?
The 96 AFA-TUBE TPX Plate has been optimized for use with the LE220-plus. The LE220-plus automatic water sensing and adjustments are key for ensuring the proper water level for the cavitation sites on the 96 AFA-TUBE TPX Plate for reproducible processing.
What sample volume can be used with the 96 AFA-TUBE TPX Plate?
Sample volumes for the 96 AFA-TUBE TPX Plate for DNA shearing can range from 5 to 50 ul. Max volume allowance is 200 μl.
Which on-deck thermocycler has the 96 AFA-TUBE TPX Plate been validated?
The 96 AFA-TUBE TPX Plate has been validated with the Applied Biosystems Automated Thermal Cycler (ATC).
Which on-deck magnetic rack has the 96 AFA-TUBE TPX Plate been validated with?
The 96 AFA-TUBE TPX Plate has been validated with the ALPAQUA Magnum FLX.
Do I need to use a specific Rack for processing 96 AFA-TUBE TPX Plate in a Covaris LE220-plus?
Yes, the Rack 96 AFA-TUBE TPX Plate (PN 500588) is required for processing the 96 AFA-TUBE TPX Plate.
Is it normal that the wells of the 96 AFA-TUBE TPX Plate are loose?
The 96 AFA-TUBE TPX Plate is designed to have a well tolerance which allows for better shearing performance. Any looseness seen in the wells of the plate is normal.